Gram-negative bacteria are the causative agents a variety of important human infectious diseases. The successful therapy of infections, caused by many of these pathogens is limited by their intrinsic resistance mechanism including the impermeable outer membrane (OM) and the activities of various efflux pumps. In this project, we propose to develop novel antibiotics that do not enter the bacterial cytoplasm, but instead, they act by interfering with the biogenesis of the OM. Two pathways, consisting of the Lol and Bam machineries, are responsible for trafficking of lipoproteins and beta-barrel non-lipidated OM proteins, respectively. These pathways are essential in Pseudomonas aeruginosa and will be targeted for disruption by small molecule inhibitors. Strains of P. aeruginosa were engineered that allow regulation of the key components of the Bam and Lol pathways and carry a luciferase reporter construct responsive to Lol and Bam depletion. These P. aeruginosa test strains will be used to screen compound libraries and inhibitors of OM protein trafficking will be identified. The compounds will be characterized to identify those with maximal killing potency potent, exhibit a broad spectrum against other Gram-negative pathogens, are active in biofilms, serum and respiratory mucus, exhibit low cytotoxicity and potentiate the bactericidal activities of other antibiotics. The protein targets of these active compounds will be indentified using genetic and chemical approaches. The efficacy of each of these compounds alone, or in combination with other antibiotics, in protecting mice against P. aeruginosa colonization will be tested in a murine respiratory infection model. This work could lead to the development of a new class of broad-spectrum inhibitors suitable for therapy of a variety of infections caused by antibiotic- resistant Gram-negative pathogens.